For most analysis based on biochip or nanoparticles, a scanner using laser induced fluorescence is widely used, since a conjugation reaction between DNA and a protein alone cannot provide an electric signal. Thus, for such case, a sample to be determined is first conjugated to a fluorescent material and then reacted with an arrayed biomaterial so that the degree of biochemical reaction can be determined by measuring fluorescence at a conjugation site. However, such fluorescence detection method is disadvantageous in that an expensive laser must be employed and it cannot be applied to an ultrafine array system.
Recently, a detection method using semi-conducting and water-soluble quantum dots such as CdSe, which emits fluorescence, has been developed instead of using fluorescence materials.
In general, a fluorescent material consisting of organic compounds is chemically unstable and its fluorescence activity is induced only when it is excited with laser having specific wavelength. On the other hand, nanoparticles (i.e., quantum dots) can be easily excited without laser so that it requires a rather simple detection instrument. In addition, it has a highly-sensitive analytical power and an excellent light stability and photoluminescence (PL) of which intensity can be adjusted. As such, as a new kind of fluorescent marker in bio-imaging field, nanoparticles are now getting more attention than ever.
Furthermore, since a biosensor using magnetic beads, magnetic nanoparticles or magnetic sensor is small and light, consumes little electricity and can be integrated, a cheap sensor can be reproducibly mass-produced. In addition, it has a high sensitivity and reliability for detecting a sample with extremely small amount. However, a difficulty to produce a complex having conjugation between magnetic nanoparticles and a specific bio-material remains as a problem for its actual application.
Although many studies have been made on synthesis of fluorescent nanoparticles or magnetic nanoparticles, there are only a few studies regarding chemical properties of conjugation between nanoparticles (or quantum dots) and bio-materials such as protein extract, antibody, and nucleotide.
Until now, many efforts have been made to identify a ligand molecule that is conjugated to nanoparticles. Specifically, nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopy, and Fourier transformed infrared spectroscopy (FT-IR), etc. were used to determine the progress of ligand substitution and bio-conjugation. However, such macroscopic analytical methods are not sufficient to identify a ligand which is actually coordinated to nanoparticles, and not suitable to determine the actual amount and the distribution of the conjugation, etc.
According to the present invention, by using TOF-SIMS, especially an imaging technology of TOF-SIMS, a method which allows an evaluation of conjugation between materials, a progress of conjugation reaction, and a quantitative analysis of conjugated materials, is provided.
Secondary ion mass spectrometry (SIMS) has been generally used as a means for obtaining depth profile of a specific impurity or its lateral profile on same surface at production level for preparing semiconductor device, as it is described in Japanese Patent Application Laid-open No. 1995-153808.
Recently, a method of analyzing biological sample by using mass spectrometry, in particular matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), has been described in Korean Patent Application Laid-open No. 2005-0106323, Japanese Patent Application Laid-open No. 2004-251623 and US Patent Application Laid-open No. 2007-0249060, respectively. However, it is used only for analyzing a product obtained after completion of a reaction, and is different from the method of the present invention wherein conjugation between two materials present on nanoparticles is determined based on a measurement result of TOF-SIMS.